Technical Abstract:
Sensitive and accurate detection is a prerequisite for efficient management and regulatory responses to prevent the introduction and spread of HLB-associated “Candidatus Liberibacter species to unaffected areas. To improve the current detection limit of HLB-associated “Ca. Liberibacter” spp, we developed a novel ultra-sensitive dual-primer TaqMan PCR for HLB molecular diagnosis. This new detection method significantly improves the sensitivity of detection of HLB-associated “Ca. Liberibacter asiaticus”. This system uses two sets of primers and two sequential amplification steps analogous to the standard nested PCR. However, unlike two-tube nested PCR, this dual-primer Taq-Man PCR is carried out in a single closed tube. The sensitivity of this dual primer detection system is significantly higher than that of a standard Taq-Man PCR and has comparable sensitivity to a two-tube nested PCR. However, the standard two-tube nested PCR procedure requires a second amplification from the first amplified product in a separate tube. This processing of the previously amplified products could cause the cross contamination and lead false positives, making this approach risky for practical application unless extreme caution is taken. In a recent survey from HLB infected citrus groves, the comparative detection rate was ~12% higher using the single-tube dual primer Taq-Man PCR protocol than that using the single primer pair real-time PCR method. Cloning and re-sequencing of the amplicons confirmed the detections were true positives. Availability of a simple, ultra-sensitive detection system will be an extremely useful tool for detecting and monitoring the spread of HLB-associated “Ca. Liberibacter”, and for early diagnosis of HLB. It is also a powerful tool for clinical testing of imported materials, monitoring vectors, and performing surveys in areas that are still free of the disease.